Name: pre-conditioning the airways of mice with bleomycin increases the efficiency of orthotopic lung cancer cell engraftment
Uploaded: 2018-06-28T12:30:00
Description: Watch this Scientific Journal Video about Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment at JoVE.com

这项研究的经费来自国家癌症研究所 (R01CA166376 和 R01CA191489 D.X.) 和国防部 (W81XWH-16-1-0227 D.X. 阮) 的赠款。

所有实验都是按照耶鲁大学机构动物护理和使用委员会 (IACUC) 批准的协议进行的。
1. 试剂的设置/准备。
<ol><li>
博莱 霉 素 注意: 根据全球统一的化学品分类和标签制度, 博莱霉素被归类为 GHS08 的健康危害。<ol>
<li>用化学罩准备博莱霉素。并用重悬 15 U 入5毫升无菌磷酸盐缓冲盐水 (PBS)。</li>
		<li>整除100-200 µL 的溶液成玻璃瓶, 并冻结在-20 摄氏度, 以供将来使用...

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2015.">Cancer statistics, 2015.</a> CA-Cancer J Clin. 65, 5-29 (2015).</li><li>Gaspar, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+metastases+in+lung+cancer.">Brain metastases in lung cancer.</a> Expert Rev Anticanc. 4, 259-270 (2004).</li><li>Hess, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+patterns+in+adenocarcinoma.">Metastatic patterns in adenocarcinoma.</a> Cancer. 106, 1624-1633 (2006).</li><li>Hoffman, P. C., Mauer, A. M., Vokes, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer.">Lung cancer.</a> Lancet. 355, 479-485 (2000).</li><li>Travis, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+lung+cancer.">Pathology of lung cancer.</a> Clin Chest Med. 23 (1), 65-81 (2002).</li><li>Chen, Z., Fillmore, C. M., Hammerman, P. S., Kim, C. F., Wong, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-small-cell+lung+cancers:+a+heterogeneous+set+of+diseases.">Non-small-cell lung cancers: a heterogeneous set of diseases.</a> Nat Rev Cancer. 14, 535-546 (2014).</li><li>Kim, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+human+non-small-cell+lung+cancer:+raising+the+bar.">Mouse models of human non-small-cell lung cancer: raising the bar.</a> Cold Spring Harb Sym. 70, 241-250 (2005).</li><li>Meuwissen, R., Berns, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+human+lung+cancer.">Mouse models for human lung cancer.</a> Gene Dev. 19, 643-664 (2005).</li><li>Junttila, M. R., de Sauvage, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+tumour+micro-environment+heterogeneity+on+therapeutic+response.">Influence of tumour micro-environment heterogeneity on therapeutic response.</a> Nature. 501, 346-354 (2013).</li><li>Nguyen, L. V., Vanner, R., Dirks, P., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+an+evolving+concept.">Cancer stem cells: an evolving concept.</a> Nat Rev Cancer. 12, 133-143 (2012).</li><li>Minchinton, A. I., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+penetration+in+solid+tumours.">Drug penetration in solid tumours.</a> Nat Rev Cancer. 6, 583-592 (2006).</li><li>Leblond, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+cell+therapy+techniques+for+respiratory+disease:+intratracheal+delivery+of+genetically+engineered+stem+cells+in+a+murine+model+of+airway+injury.">Developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury.</a> Hum Gene Ther. 20, 1329-1343 (2009).</li><li>Vaughan, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-negative+progenitors+mobilize+to+regenerate+lung+epithelium+after+major+injury.">Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury.</a> Nature. 517, 621-625 (2015).</li><li>Zuo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p63(+)Krt5(+)+distal+airway+stem+cells+are+essential+for+lung+regeneration.">p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration.</a> Nature. 517, 616-620 (2015).</li><li>Mahoney, J. E., Kim, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+potential+of+lung+progenitors.">Tracing the potential of lung progenitors.</a> Nat Biotechnol. 33, 152-154 (2015).</li><li>Wang, D., Morales, J. E., Calame, D. G., Alcorn, J. L., Wetsel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+embryonic+stem+cell-derived+alveolar+epithelial+type+II+cells+abrogates+acute+lung+injury+in+mice.">Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.</a> Mol Ther. 18, 625-634 (2010).</li><li>Ortiz, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+engraftment+in+lung+is+enhanced+in+response+to+bleomycin+exposure+and+ameliorates+its+fibrotic+effects.">Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects.</a> Proc Natl Acad Sci USA. 100, 8407-8411 (2003).</li><li>Suzuki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+the+size+of+central+fibrosis+in+peripheral+adenocarcinoma+of+the+lung.">Prognostic significance of the size of central fibrosis in peripheral adenocarcinoma of the lung.</a> Ann Thorac Surg. 69, 893-897 (2000).</li><li>Cancer Genome Atlas Research, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+profiling+of+lung+adenocarcinoma.">Comprehensive molecular profiling of lung adenocarcinoma.</a> Nature. 511, 543-550 (2014).</li><li>Scotton, C. J., Chambers, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleomycin+revisited:+towards+a+more+representative+model+of+IPF?.">Bleomycin revisited: towards a more representative model of IPF?.</a> Am J Physiol-Lung C. 299, L439-L441 (2010).</li><li>Hay, J., Shahzeidi, S., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+bleomycin-induced+lung+damage.">Mechanisms of bleomycin-induced lung damage.</a> Arch Toxicol. 65, 81-94 (1991).</li><li>Vaccaro, C. A., Brody, J. S., Snider, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+wall+basement+membranes+in+bleomycin-induced+pulmonary+fibrosis.">Alveolar wall basement membranes in bleomycin-induced pulmonary fibrosis.</a> Am Rev Respir Dis. 132, 905-912 (1985).</li><li>Venkatesan, N., Ebihara, T., Roughley, P. J., Ludwig, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+large+and+small+proteoglycans+in+bleomycin-induced+pulmonary+fibrosis+in+rats.">Alterations in large and small proteoglycans in bleomycin-induced pulmonary fibrosis in rats.</a> Am J Resp Crit Care. 161, 2066-2073 (2000).</li><li>Moore, B. B., Hogaboam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+pulmonary+fibrosis.">Murine models of pulmonary fibrosis.</a> Am J Physiol-Lung C. 294, L152-L160 (2008).</li><li>Izbicki, G., Segel, M. J., Christensen, T. G., Conner, M. W., Breuer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+bleomycin-induced+lung+fibrosis.">Time course of bleomycin-induced lung fibrosis.</a> Int J Exp Pathol. 83, 111-119 (2002).</li><li>Schrier, D. J., Phan, S. H., McGarry, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+the+nude+(nu/nu)+mutation+on+bleomycin-induced+pulmonary+fibrosis.+A+biochemical+evaluation.">The effects of the nude (nu/nu) mutation on bleomycin-induced pulmonary fibrosis. A biochemical evaluation.</a> Am Rev Respir Dis. 127, 614-617 (1983).</li><li>Ponomarev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+triple-modality+reporter+gene+for+whole-body+fluorescent,+bioluminescent,+and+nuclear+noninvasive+imaging.">A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging.</a> Eur J Nucl Med Mol I. 31, 740-751 (2004).</li><li>Morten, B. C., Scott, R. J., Avery-Kiejda, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Three+Different+Methods+for+Determining+Cell+Proliferation+in+Breast+Cancer+Cell.">Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell.</a> J. Vis. Exp. , (2016).</li><li>Tseng, J. C., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+bioluminescence+imaging+of+mouse+tumor+models.">Quantitative bioluminescence imaging of mouse tumor models.</a> Cold Spring Harbor protocols. , (2015).</li><li>Byrne, F. L., McCarroll, J. A., Kavallaris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+Tumor+Burden+In+Vivo+and+Metastasis+Ex+Vivo+Using+Luciferase-Expressing+Cancer+Cells+in+an+Orthotopic+Mouse+Model+of+Neuroblastoma.">Analyses of Tumor Burden In Vivo and Metastasis Ex Vivo Using Luciferase-Expressing Cancer Cells in an Orthotopic Mouse Model of Neuroblastoma.</a> Methods Mol Biol. 1372, 61-77 (2016).</li><li>Parkinson, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+necropsy+and+selected+tissue+and+sample+collection+in+rats+and+mice.">Diagnostic necropsy and selected tissue and sample collection in rats and mice.</a> J. Vis. Exp. , (2011).</li><li>Tammela, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wnt-producing+niche+drives+proliferative+potential+and+progression+in+lung+adenocarcinoma.">A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma.</a> Nature. 545, 355-359 (2017).</li><li>DuPage, M., Dooley, A. L., Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+mouse+lung+cancer+models+using+adenoviral+or+lentiviral+delivery+of+Cre+recombinase.">Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase.</a> Nat Protoc. 4, 1064-1072 (2009).</li><li>Byrne, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+open+issues+in+cancer+precision+medicine+with+patient-derived+xenografts.">Interrogating open issues in cancer precision medicine with patient-derived xenografts.</a> Nat Rev Cancer. 17, 254-268 (2017).</li><li>Nguyen, D. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WNT/TCF+signaling+through+LEF1+and+HOXB9+mediates+lung+adenocarcinoma+metastasis.">WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis.</a> Cell. 138, 51-62 (2009).</li><li>Raghu, G., Nyberg, F., Morgan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+interstitial+lung+disease+and+its+association+with+lung+cancer.">The epidemiology of interstitial lung disease and its association with lung cancer.</a> Brit J Cancer. 91, S3-S10 (2004).</li><li>Hubbard, R., Venn, A., Lewis, S., Britton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer+and+cryptogenic+fibrosing+alveolitis.+A+population-based+cohort+study.">Lung cancer and cryptogenic fibrosing alveolitis. A population-based cohort study.</a> Am J Resp Crit Care. 161, 5-8 (2000).</li><li>Nagai, A., Chiyotani, A., Nakadate, T., Konno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer+in+patients+with+idiopathic+pulmonary+fibrosis.">Lung cancer in patients with idiopathic pulmonary fibrosis.</a> Tohoku J Exp Med. 167, 231-237 (1992).</li><li>Rock, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+stromal+populations+contribute+to+pulmonary+fibrosis+without+evidence+for+epithelial+to+mesenchymal+transition.">Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition.</a> Proc Natl Acad Sci USA. 108, E1475-E1483 (2011).</li><li>Saito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+after+surgery+for+pathologic+stage+IA+non-small+cell+lung+cancer+associated+with+idiopathic+pulmonary+fibrosis.">Survival after surgery for pathologic stage IA non-small cell lung cancer associated with idiopathic pulmonary fibrosis.</a> Ann Thorac Surg. 92, 1812-1817 (2011).</li><li>Stevens, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+Receptor+Expression+in+Subtypes+of+Lung+Adenocarcinoma+Potentiates+Outgrowth+of+Micrometastases.">Extracellular Matrix Receptor Expression in Subtypes of Lung Adenocarcinoma Potentiates Outgrowth of Micrometastases.</a> Cancer Res. 77, 1905-1917 (2017).</li><li>Harrison, J. H., Lazo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dose+continuous+infusion+of+bleomycin+in+mice:+a+new+model+for+drug-induced+pulmonary+fibrosis.">High dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fibrosis.</a> J Pharmacol Exp Ther. 243, 1185-1194 (1987).</li><li>Shcherbo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+far-red+fluorescent+protein+for+whole-body+imaging.">Bright far-red fluorescent protein for whole-body imaging.</a> Nature Methods. 4, 741-746 (2007).</li><li>Aso, Y., Yoneda, K., Kikkawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+biochemical+study+of+pulmonary+changes+induced+by+bleomycin+in+mice.">Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice.</a> Lab Invest. 35, 558-568 (1976).</li><li>Kim, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+bronchioalveolar+stem+cells+in+normal+lung+and+lung.">Identification of bronchioalveolar stem cells in normal lung and lung.</a> Cell. 121, 823-835 (2005).</li><li>Fichtner, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+patient-derived+non-small+cell+lung+cancer+xenografts+as+models+for+the+identification+of+predictive+biomarkers.">Establishment of patient-derived non-small cell lung cancer xenografts as models for the identification of predictive biomarkers.</a> Clin Cancer Res. 14, 6456-6468 (2008).</li><li>Zhang, X. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+patient-derived+non-small+cell+lung+cancer+xenograft+models+with+genetic+aberrations+within+EGFR,+KRAS+and+FGFR1:+useful+tools+for+preclinical+studies+of+targeted+therapies.">Establishment of patient-derived non-small cell lung cancer xenograft models with genetic aberrations within EGFR, KRAS and FGFR1: useful tools for preclinical studies of targeted therapies.</a> J Transl Med. 11, 168 (2013).</li></ol>

在肺癌和其他慢性疾病的肺36之间有明显的临床相似之处。尤其是特发性肺纤维化 (IPF) 患者对发展肺癌的偏爱增加, 而这个协会独立于吸烟史37,38。森林小组的特点是逐步破坏肺结构和受损的呼吸功能, 通过沉积 ECM39。同时, 在手术切除后, 早期的无细胞肺癌患者并发森林小组的结局较差40。多数 NSCLC ?...

肺癌是全球1癌症相关死亡的主要原因。肺癌患者最终从转移到远端器官, 主要是中枢神经系统, 肝脏, 肾上腺, 骨骼2,3,4。胸恶性肿瘤历来被归类为小细胞肺癌或非小细胞肺癌 (NSCLC)5。NSCLC 是最常见的恶性肿瘤, 可分为不同的组织学亚型, 包括肺腺癌 (LUAD) 和肺鳞状细胞癌 (LUSC)6。经手术切除的人原发性肺癌的基因组分析显示, 某一 histotype 内的肿瘤也能表达多种分子扰动, 进一步促进其临床进展和混淆患者预后。肺癌的显著异质性对合理的设计、临床前试验和有效的治疗策略的实施都是一个重大挑战。因此, 有必要扩大可听话的实验性肺癌模型的汇辑, 以研究这种疾病的不同细胞来源、分子亚型和分期。
使用动物模型的各种方法在体内研究肺癌, 每个都有各自的优缺点, 这取决于所关心的生物学问题。基因工程小鼠模型 (GEMMs) 可以针对特定祖细胞类型的特异基因改变, 导致肿瘤在免疫宿主7中取得进展。虽然非常强大和临床相关的, 潜伏期, 变异性和/或肺部肿瘤发病率与 GEMMs 可能会禁止某些定量测量和检测晚期转移在遥远的器官8。一个补充的方法是使用同种异体模型, 藉以肺癌细胞从老鼠肿瘤直接获得或首先被获取作为建立的细胞线在文化中被重新介绍入自体寄主。类似, 肺癌移植是由人体细胞系或病人衍生的肿瘤样本建立的。人细胞系移植或患者衍生的移植 (PDXs) 一般维持在免疫缺陷小鼠, 因此排除完全免疫监测9。尽管有这个缺点, 它们提供了一个途径来传播限制数量的人类 biospecimens 和研究人类癌细胞的基本的体内性质, 这编码为更复杂的基因组畸变比 GEMM 肿瘤。
同种异体移植和异种移植的一个有用的特性是, 他们可以使用传统的限制性细胞稀释方法, 用于量化10恶性细胞中肿瘤起始细胞 (抽搐) 的频率。在这些实验中, 在动物的侧面注入了一定数量的细胞, 并根据肿瘤的发生率来估计抽搐频率。然而皮下肿瘤可以是更低氧11并且可能不模型主要生理制约肺肿瘤微环境。气管内向小鼠肺内输送上皮干细胞或祖细胞是研究肺再生和气道干细胞生物学12的方法。然而, 这种技术的植入率可能相对较低, 除非肺部首先受到生理形式的伤害, 如病毒感染13,14。炎症基质细胞的支持和 (或) 肺基底膜的破坏, 可改善移植细胞在远端气道15的相关干细胞龛位的保留。纤维化诱导剂也可以预条件肺, 以增强植入诱导多能细胞16和间充质干细胞17。类似的气道损伤形式是否会影响植入率, 肿瘤的启动能力和肺癌细胞的产生还有待系统地评估。
本文通过对损伤小鼠肺的预处理, 描述了提高原位肺癌细胞植入效率的方法。LUAD 出现在远端呼吸道与这些癌症的一个重要的子集发展纤维间质基质18 , 往往与不良预后19。博莱霉素是一种天然非核糖体杂交肽-聚酮体, 已广泛用于诱导小鼠肺纤维化20。博莱霉素的气道灌注首先促进肺泡上皮的磨损和炎症细胞的招募, 包括巨噬细胞、中性粒细胞和单核细胞21。其次是在远端呼吸道组织重塑, 基底膜重组22,23和细胞外基质 (ECM) 沉积24。单个博莱霉素注射液的影响是瞬变的, 在大多数研究中30天后纤维化解决25。利用同种异体移植和异种移植模型, 我们对博莱霉素小鼠呼吸道进行预适应试验, 可以显著提高肺 LUAD 细胞的服用率。

<table>
	<tbody>
		<tr>
			<td>Bleomycin</td>
			<td>Sigma</td>
			<td>B5507-15UN</td>
			<td>CAUTION Health hazard GHS08</td>
		</tr>
		<tr>
			<td>Exel Catheter 24G</td>
			<td>Fisher</td>
			<td>1484121</td>
			<td>Remove needle. For intratracheal injection</td>
		</tr>
		<tr>
			<td>Ketamine (Ketaset inl 100 mg/mL C3N 10 mL)</td>
			<td>Butler Schein</td>
			<td>56344</td>
			<td>To anesthetize mice</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Butler Schein</td>
			<td>33198</td>
			<td>To anesthetize mice</td>
		</tr>
		<tr>
			<td>Ketoprofen, 5,000 mg</td>
			<td>Cayman Chemical</td>
			<td>10006661</td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Puralube Veterinary Ophthalmic Ointment</td>
			<td>BUTLER ANIMAL HEALTH COMPANY LLC</td>
			<td>8897</td>
			<td>To prevent eye dryness while under anesthesia</td>
		</tr>
		<tr>
			<td>D-Luciferin powder</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>122799</td>
			<td>For luminescent imaging. Reconstitute powder with PBS for a working concentration of 15mg/mL. Protect from Light</td>
		</tr>
		<tr>
			<td>Rodent Intubation stand</td>
			<td>Braintree Scientific</td>
			<td>RIS-100</td>
			<td>Recommended stand for intratracheal injection</td>
		</tr>
		<tr>
			<td>MI-150 ILLUMINATOR 150W MI-150</td>
			<td>DOLAN-JENNER INDUSTRIES</td>
			<td>MI-150 / EEG2823M</td>
			<td>To illuminate and visualize trachea</td>
		</tr>
		<tr>
			<td>Graefe Forceps, 2.75 (7 cm) long serrat</td>
			<td>Roboz</td>
			<td>RS-5111</td>
			<td>For intratracheal injection</td>
		</tr>
		<tr>
			<td>Syringe Luer-Lok Sterile 5ml</td>
			<td>BD / Fisher</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Satiny Smooth by Conair Dual Foil Wet/Dry Rechargeable Shaver</td>
			<td>Conair</td>
			<td>-</td>
			<td>To shave mice</td>
		</tr>
		<tr>
			<td>Bonn Scissors, 3.5&#34; straight 15 mm sharp/sharp sure cut blades</td>
			<td>Roboz</td>
			<td>RS-5840SC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>BD / Fisher</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>USA SCIENTIFIC INC</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial Scintillation 7 mL Borosilicate Glass GPI</td>
			<td>Fisher</td>
			<td>701350</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter pipette tips (200 &#956;L)</td>
			<td>USA SCIENTIFIC INC</td>
			<td>1120-8710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum USDA</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A2942-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Flask T/C 75cm sq canted neck, blue cap</td>
			<td>Fisher / Corning</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum Xenogen Bioluminiscence</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>124262</td>
			<td>For in vivo bioluminescence imaging</td>
		</tr>
		<tr>
			<td>Living image software</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>128113</td>
			<td>For in vivo bioluminescence analysis</td>
		</tr>
		<tr>
			<td>XGI-8 Gas Anesthesia System</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>118918</td>
			<td>For Isoflurane anesthesia</td>
		</tr>
		<tr>
			<td>BD Ultra-Fine II Short Needle Insulin Syringe 1 cc. 31 G x 8 mm (5/16 in)</td>
			<td>BD / Fisher</td>
			<td>BD328418</td>
			<td>For retro-orbital luciferin injection</td>
		</tr>
		<tr>
			<td>Syringe 1ml</td>
			<td>BD / Fisher</td>
			<td>14-823-434</td>
			<td>For intraperitoneal injections</td>
		</tr>
		<tr>
			<td>26 G x 1/2 in. needle</td>
			<td>BD / Fisher</td>
			<td>305111</td>
			<td>For intraperitoneal injections</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>VWR</td>
			<td>43368-9M</td>
			<td>CAUTION Health hazard GHS07, GHS08. For fixing tissue</td>
		</tr>
		<tr>
			<td>Pipet-Lite Pipette, Unv. SL-200XLS+</td>
			<td>METTLER-TOLEDO INTERNATIONAL</td>
			<td>17014411</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s Hematoxylin</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>517-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y stain 0.25% (w/v) in 57%</td>
			<td>Fisher</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson Trichrome Stain Kit</td>
			<td>IMEB Inc</td>
			<td>K7228</td>
			<td>For masson trichrome stain to visualize collagen</td>
		</tr>
		<tr>
			<td>Superfrost plus glass slides</td>
			<td>Fisher</td>
			<td>1255015</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>C3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Mycoplasma Detection Kit</td>
			<td>ATCC</td>
			<td>30-1012K</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Embedding compound</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>62550-12</td>
			<td>For embedding tissue for frozen sections</td>
		</tr>
		<tr>
			<td>Leica CM3050 S Research Cryostat</td>
			<td>Leica</td>
			<td>CM3050 S</td>
			<td>To section tissue for staining analysis</td>
		</tr>
		<tr>
			<td>Keyence All-in One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X700</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>US National Institutes of Health</td>
			<td>IJ1.46</td>
			<td>http://rsbweb.nih.gov/ij/ download.html</td>
		</tr>
		<tr>
			<td>Prism 7.0 for Mac OS X</td>
			<td>GraphPad Software, Inc.</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Athymic (Crl:NU(NCr)-Foxn1nu) mice</td>
			<td>Charles River</td>
			<td>NIH-553</td>
			<td></td>
		</tr>
		<tr>
			<td>NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice</td>
			<td>Jackson Laboratories</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr>
			<td>B6129SF1/J mice</td>
			<td>Jackson Laboratories</td>
			<td>101043</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH-H2030 cells</td>
			<td>ATCC</td>
			<td>CRL-5914</td>
			<td></td>
		</tr>
		<tr>
			<td>368T1</td>
			<td>generously provided by Monte Winslow (Standford University)</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>PC9 cells</td>
			<td>Nguyen DX et al. Cell. 2009;138:51&#8211;62</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>H2030 BrM3 cells</td>
			<td>Nguyen DX et al. Cell. 2009;138:51&#8211;62</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

pre-conditioning the airways of mice with bleomycin increases the efficiency of orthotopic lung cancer cell engraftment

肺癌是一种致命的治疗顽固性疾病, 是生物异构的。为了了解和有效地治疗胸恶性肿瘤的完整临床谱, 需要额外的动物模型来重述不同的人肺癌亚型和分期。同种异体或异种移植模型是多种多样的, 可以利用小鼠或人类的恶性细胞,在体内癌变能力的量化。然而, 以前所描述的肺癌细胞植入的方法已经在非生理部位进行, 如小鼠的侧面, 由于细胞移植到肺部的效率低下。在本研究中, 我们描述了一种通过对小鼠肺纤维化诱发博莱霉素进行预调节来增强原位肺癌细胞植入的方法。作为一种概念验证实验, 我们将这种方法应用于从老鼠或人源获得的肺腺癌亚型的嫁接肿瘤细胞, 并将其转化为不同品系的小鼠。我们证明, 在肿瘤细胞注射前, 与博莱霉素的呼吸道损伤增加了肿瘤细胞从0-17% 到71-100% 的植入。显著地, 该方法通过不同模型和小鼠菌株增强肺肿瘤发生率和后续生长。此外, 嫁接肺癌细胞从肺部传播到相关的远端器官。因此, 我们提供了一个协议, 可用于建立和维持新的肺癌原位模型, 限制数量的细胞或 biospecimen, 并定量评估肺癌细胞在生理相关设置的癌变能力.

为了提高 LUAD 癌细胞植入到小鼠肺中的效率, 我们制定了一项协议, 首先使用博莱霉素的呼吸道, 其次是原位肿瘤细胞注射 (图 1)。我们证实, 即使在免疫裸小鼠, 博莱霉素诱导瞬变纤维化14天, 证明了气道结构的损失和增加胶原沉积 (图 2)。这些小鼠的毛纤维化在博莱霉素注射液后50天解决 (图 2; 右面板) 与以...

Watch this Scientific Journal Video about Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment at JoVE.com

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

本文介绍一种通过对气道损伤进行预适应来显著提高肺癌细胞原位植入的方法。这种方法也可以应用于研究肺微环境中的间质相互作用, 转移传播, 肺癌的共同发病, 并更有效地产生病人的移植。

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

The overall goal of this procedure is to increase the efficiency of transplanting tumor-initiating lung cancer cells into the airways of mice. This method can help establish new preclinical models in order to answer key questions in cancer biology, including the causes of drug resistance and relationship between fibrosis and lung cancer. The main advantage of this technique is that it increase the transplantation efficiency of tumor-initiating cells into the lungs of a variety of mouse strains.The implications of this technique extend toward therapy, because it enables us to model a greater variety of human lung cancer subtypes, known to have different clinical responses to treatment. For this protocol, the mice must first be injected with bleomycin. Then, after 14 days, the tumor cells are injected.The procedure for both injections is very similar and described here. Two hours prior to the injection, thaw the bleomycin stock on ice and dilute it to the working concentration. Keep it on ice.For a cell bolus, have a cell suspension prepared as described in the text protocol. The procedure time for each mouse is about 10 minutes all together. Be certain to confirm proper anesthesia using a toe pinch, and apply vet ointment to the eyes.At this time, if the mouse has dark hair, then shave the entire rib area both ventrally and dorsally so it can be imaged correctly. Then, place the mouse on the intubation platform by securing it by its front teeth with it's back against the platform. If a cell bolus is to be injected, resuspend it now with gentle trituration.Now illuminate the upper-chest region, open the mouth, and gently pull out the tongue using flat forceps. Prepare an IV catheter for the trachea by removing the needle, then position it over the tracheal opening. Next, insert the catheter into the trachea until the top of the catheter is near the front teeth and illuminate the interior of the catheter to confirm its placement.Then, dispense the 50 microliters of freshly made bleomycin, or cell suspension, directly into the catheter. Wait a few seconds for the entire volume to travel down the catheter. Then, remove the catheter and dispose of it in 10%bleach.If the mouse is not inhaling the liquid, carefully monitor its breathing and adjust the catheter position. If the mouse stops breathing, immediately remove the catheter and allow the mouse to breath before you reinsert the catheter in. After removing the catheter, continue performing the procedure to have five treated mice, still anesthetized, and ready to image.For recovery, place the mouse supine on an IACUC approved heating pad in a recovery cage and monitor it until it has fully recovered. Two or three minutes after injecting the cells, inject 100 microliters of luciferin retro-orbitally, using an insulin needle. Alternate the eye used for injection every imaging session.After waiting at lest two minutes, position up to five mice in an animal bioluminescence imager, in the sternal recumbency position and acquire a dorsal picture using luminescence settings. Under the control panel, adjust the resolution and sensitivity settings, to measure the luminescence of the cells. For most applications, start with three minutes, set the binning to medium, set the f/stop to one, set the field of view to d, and set the subject height to 1.5.If the injection was successful, a signal is detected in the upper chest area in one lung or both lungs. If cells are clustered at the entrance of the trachea, the injection was unsuccessful. After taking the dorsal image, flip the mice and acquire a ventral image and adjust the setting as needed.After imaging, proceed with postoperative care as described in the text protocol. Then, repeat this imaging protocol three days post-engraftment and weekly thereafter. To analyze the images, use a bioluminescence analysis software.First, load all the appropriate images in the series. This section is detailed to one software package. Next, for each mouse, in the first image of the series, create a square region of interest and position it over the upper chest area covering the entire lung region.Then, using a right click, access the copy all regions of interest function, and use this tool to apply the same regions of interest to each image in the group. Then, within each image, tweak the positions of the squares to cover the upper chest of each mouse. Do this for both the ventral and dorsal views.Now, in the upper left corner of the image window, select the photons function. Then select the measure option. The data from the regions of interest is then displayed in an output table as total flux.Analyze this data as needed. When immune compromised, athymic mice were treated with bleomycin. Prior to cell-bolus engraftment, transient fibrosis was observed by day 14 in bleomycin-treated mice, but not vehicle-treated mice.Next, cells from the established human lung cancer cell line, H2030, were delivered intratracheally into the lungs of these mice. After 35 days, bleomycin-treated mice had a high lung-tumor burden. However, in vehicle treated animals, no tumors were detected.The lung-tumor burden was confirmed using histology. Lungs pretreated with vehicle had no evidence of tumor nodules, whereas bleomycin pretreated lungs had large tumor nodules. After transplanting a metastatic H2030 BrM 3 cell line, by 39 to 57 days, bioluminescence was too intense for imaging, so metastasis was analyzed in distant organs ex vivo.Small lesions could be detected in various tissues, suggesting that this method holds promise, to study spontaneously disseminated disease. Once mastered, this technique can be done in 10 minutes per cage of five mice without imaging and 20 minutes per cage when also imaging the mice. While attempting this procedure, it is important to remember to ensure proper insertion of the catheter and to monitor the animal's breathing.Following this procedure, other methods like pharmacological studies or aminoacetic chemistry of harvested tissue can be performed in order to answer additional questions, like how engrafted tumors respond to drugs or characterizing tumor stromal interactions specific to the lungs. After its development, this technique can be applied to different mouse strains and tumor models, including patient-derived xenografts in a physiologically relevant setting. Don't forgot that working with bleomycin can be extremely hazardous and precautions such as manipulation in a biohazard hood and proper disposal should always be taken while performing this procedure.

Research

JoVE Journal

Medicine

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

支气管Thermoplasty：严重哮喘的一种新的治疗方法

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

科研

医学

An Orthotopic Model of Murine Bladder Cancer

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

心导​​管在小鼠测量压力体积的关系：调查的影响鲍迪奇

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

“以病人为像”肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...